The present invention relates in general to nucleic acids an more particularly to methods for mapping expressed nucleic acid sequences to their chromosomal location, and for mapping gene response patterns.
Mapping expressed sequences to their chromosomal locations can be an important tool in the effort to use genetics to understand the basis of disease in plants and animals, e.g., humans. Two commonly used mapping techniques are genetic mapping and physical mapping. A gene is mapped xe2x80x9cgeneticallyxe2x80x9d when a polymorphism within the gene is shown to co-segregate with other polymorphisms through multiple generations. Physical mapping is performed by the demonstration of physical hybridization of a DNA probe from the gene to a chromosome (such as with fluorescent in situ hybridization), by direct DNA sequencing of a genomic region of known chromosomal location, or by the amplification of a piece of a gene from a panel of cell lines that each bear a portion of a genome of interest. See, e.g., Trask 1991 Trends Genet. 7: 149-154; Gyapay 1996 Hum. Mol. Genet. 5: 339-346; Lunetta et al., 1994 Genomics 21: 92-103; Priat et al., 1998 Genomics 54: 361-378; Steen, et al. Genome Res. Online (published online May 21, 1999); Watanabe, et al. 1999 Nat. Genet. 22: 27-36; Khan, et al. 1992 Nat. Genet. 2: 180-185; and Walter, et al. 1994 Nat. Genet. 7: 22-28.
One type of physical mapping includes performing gene-specific PCR reactions on a panel of somatic cell or radiation hybrids. These hybrids include cells having, in addition to their endogenous genome, DNA from a second species. The pattern of the presence or absence of a product can be compared to patterns of markers whose physical location is known, thus assigning a fairly accurate physical location to the gene of interest. Efforts to map large numbers of genes using radiation hybrid panels have been somewhat successful, but a large failure rate, sometimes as large as 30% or more, results from the presence of introns in genomic DNA. The introns prevent some primer pairs designed from cDNA from amplifying their target DNA. In addition, either a preliminary physical map or a large amount of genetic data is necessary to begin mapping genes using this method.
Thus, a need remains in the art for more viable method of mapping large numbers of expressed sequences to their chromosomal locations. Such methods would greatly facilitate analysis of the genetic basis for gene expression and its relationship to diseases in all types of organisms.
The present invention provides method for determining the chromosomal location of a nucleic acid sequence. Also provided are methods for identifying at least one gene whose expression is modulated by one or more exogenous genes present in a hybrid cell, and a method of mapping a network response.
This new method of mapping genes has utility beyond human genes, the majority of which will likely be mapped in the next year to two years. Canine, porcine, zebrafish, and many other genomes have neither been well-mapped, nor is there a large expressed sequence database for these organisms, yet there are valuable genetic models in these species and others which could be utilized. In these organisms and others, the application of this technique to a radiation hybrid panel, as opposed to somatic cell hybrids, could increase the number and accuracy of genes mapped.
The success of this method in identifying the map locations of expressed sequences is dependent upon the ability of the host cell to permit the proper transcription and splicing of genes from the exogenous DNA. Generally, only a fraction of genes on the exogenous DNA will be transcribed in a given cell type. xe2x80x9cIllegitimate transcription,xe2x80x9d however, can be induced by agents such as cycloheximide and may significantly increase the fraction of genes that can be mapped in this way11-12. The primary difficulty in the use of this method is distinguishing between the genes expressed from the exogenous DNA and the endogenous genes that are modulated as a result of the expression of specific exogenous genes. When the complete sequence of the human genome is known, a human cell line will be the ideal vector for this protocol because the endogenous genes can be removed from the analysis by direct comparison to the human genome database. However, the identification of genes that are specifically modulated as the result of the presence of an exogenous gene or genes can potentially become a valuable functional genomics tool to build networks of gene response. The identification of which exogenous gene(s) lead to activation of endogenous gene(s) of interest through a systematic method of analyzing a large number of genetic combinations for differential gene expression may lead to a better understanding of genetic interactions. Thus, the xe2x80x9cnoisexe2x80x9d in this mapping experiment may help decode patterns of gene response on a genome-wide scale.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.